The goal of this project is to determine the mechanism by which activation of cAMP signaling pathways leads to transcriptional repression of the mouse mammary tumor virus (MMTV) promoter in the physiological context of ordered chromatin. Our previous work has shown that cAMP signaling causes repression of the MMTV promoter in a glucocorticoid-independent fashion only when it is incorporated into ordered, replicating chromatin in cultured cells. In contrast, a transiently-transfected MMTV promoter construct which does not have an organized nucleoprotein structure is activated by cAMP (cyclic AMP) signaling. These results emphasize the importance of studying transcriptional regulation of genes in their native setting of complex chromatin. We have recently determined that the two templates are regulated through different cAMP-dependent mechanisms. Thus the differences in response to cAMP signaling are not due to the same cAMP signaling cascade having two different outcomes due to the distinct structures of the templates. Rather, the structure of the template determines which cascades targets it. This model might be used to explain cell type-specific differences in regulation of the same promoter. If the chromatin structure of the promoter is in distinct configurations in two different cell types, the promoter may respond differently to the same extracellular signal. In the past year we have made significant progress in defining the mechanism by which the MMTV promoter in ordered chromatin is repressed by cAMP signaling. We approached this question through comparison to another agent which represses the promoter, the histone deacetylase inhibitor trichostatin A (TSA). We find that the mechanisms of TSA and cAMP-induced repression of the MMTV promoter are very similar. Like cAMP, TSA represses the promoter in a chromatin-specific fashion. In both cases, transcription is rapidly inhibited in a fashion independent of changes in chromatin remodeling at the template. Since the primary effect of TSA on cellular metabolism is to inhibit deacetylases and effectively raise the level of histone acetylation, both cAMP and TSA may mediate repression in part through the acetylation of factors and/or histones. We are currently developing the chromatin immunoprecipitation (ChIP) assay to examine this hypothesis. Our study of cAMP-induced repression of the MMTV promoter has led to another interesting line of investigation. In an effort to determine whether CBP was involved in this mechanism we expressed the adenovirus protein E1A in our cell lines. This protein is thought to bind CBP and inhibit its function in transcription. Unexpectedly we found that E1A has two effects on transcriptional regulation of the MMTV promoter in ordered chromatin. First, it significantly stimulates both basal and glucocorticoid-induced transcription. In addition, it alleviates repression of the promoter by cAMP. Using forms of E1A mutated at various amino acid residues necessary for binding of CBP/p300, Rb (retinoblastoma protein), and the HAT, PCAF, we have determined that CBP binding plays a role in the alleviation of cAMP-induced repression. This study provides a new avenue for understanding the mechanism of cAMP-induced repression at the MMTV promoter in chromatin.